Ropivacaine represses the ovarian cancer cell stemness and facilitates cell ferroptosis through inactivating the PI3K/AKT signaling pathway

Abstract

Purpose

Ovarian cancer is a malignant tumor in women all over the world. Ropivacaine is identified as a potential drug for the treatment of malignant tumors, but the role and mechanism of ropivacaine in ovarian cancer remains unknown.

Materials and methods

Ovarian cancer cells were treated with different doses of ropivacaine. The function of ropivacaine in ovarian cancer was assessed using Cell Counting Kit-8 assay, flow cytometry, sphere-formation assay, Western blot, Fe²⁺ level analysis, and immunofluorescence. Meanwhile, the mechanism of ropivacaine in ovarian cancer was investigated by multiple molecular experiments. The protective function of ropivacaine in ovarian cancer was further confirmed by in vivo assay.

Results

The functional research data indicated that the growth and stemness of ovarian cancer cells were restrained after ropivacaine treatment, while the ferroptosis in ovarian cancer cells was facilitated. The mechanism results confirmed that ropivacaine inactivated the PI3K/AKT signaling pathway in ovarian cancer cells. Furthermore, in vivo assay demonstrated that ropivacaine repressed the proliferation of ovarian cancer cells in vivo and had a protective function in ovarian cancer.

Conclusion

Ropivacaine restrained ovarian cancer cell stemness and accelerated cell ferroptosis by inactivating PI3K/AKT signaling pathway.

Keywords

Ovarian cancer ropivacaine stemness ferroptosis PI3K/AKT signaling pathway

Introduction

Ovarian cancer has the highest mortality rate among gynecologic tumors that threaten the health of women.¹ About 70% of the cases are diagnosed as advanced stage due to the nonspecific clinical manifestations of early ovarian cancer.² Despite the good progress that has been made in the drug treatment of ovarian cancer over the past few decades, the non-specificity and side effects of most drugs severely restrict the ovarian cancer therapeutic effect.³ Therefore, discovering more effective drugs for ovarian cancer treatment and elucidating its possible mechanism are conducive to alleviating ovarian cancer.

Cancer stem cells are a subpopulation of tumor cells with the same ability of self-renewal and differentiation as stem cells, mainly participating in tumor initiation, metastasis, and resistance to chemotherapy.⁴ Recent studies have confirmed that cancer stem cells are associated with the growth and metastasis of ovarian cancer. For example, Wilczyński et al. expounded that the existence of cancer stem cells is the main cause of chemotherapy resistance and recurrence in the treatment of ovarian cancer⁵; Muñoz-Galván et al. demonstrated that the combination treatment with cisplatin and YAP inhibitors restrain MYPT1 induced resistance, and the elevated chemotherapy resistance of ovarian cancer is accompanied by the increased stemness.⁶ These studies suggest that reducing ovarian cancer cell stemness improves ovarian cancer.

Ferroptosis is a novel form of cell death distinct from other known pathways of cell death.⁷ Ferroptosis is caused by the accumulation of lipid ROS and is characterized by cytological changes, mainly including the reduction of mitochondrial crest and rupture of the mitochondrial outer membrane.⁸ As has been reported, MAP30 protein extracted from Momordica charantia has therapeutic effects on ovarian cancer by changing metabolism and inducing ferroptosis.⁹ Thus, facilitating ferroptosis is beneficial for relieving ovarian cancer.

Ropivacaine is the most commonly used local anesthetics in clinical practice and is used clinically in surgical anesthesia and acute pain management.¹⁰ Ropivacaine has strong antitumor activity against a variety of tumor cells. For instance, ropivacaine has the function of repressing cervical cancer cell growth by suppressing cell cycle progression and accelerating cell apoptosis, prompting that ropivacaine is expected to become a therapeutic drug for clinical cancer¹¹; ropivacaine slows down the development of esophageal cancer cells by repressing the Rac1/JNK/Paxillin/FAK pathway, providing novel insights into the local anesthetics treatment for tumors.¹² However, the role of ropivacaine in ovarian cancer is not fully understood. In the current study, we preliminarily explored the ropivacaine function in ovarian cancer and confirmed that ropivacaine inhibited ovarian cancer cell growth. Aldehyde dehydrogenase (ALDH) is an enzyme that participates in the regulation of various cellular mechanisms through aldehyde detoxification.¹³ Also, the ALDH activity, sphere-formation assay, and analysis of stemness marker (OCT4 and Nanog) expressions are often used to assess the stemness of tumor cells.¹⁴ As expected, ropivacaine reduced the ALDH positive cells, weakened the sphere-formation ability of SKOV3 cells, and decreased the protein levels of OCT4 and Nanog, implying that ropivacaine restrained the stemness of ovarian cancer cells. Meanwhile, we further confirmed that ropivacaine accelerated the ferroptosis in ovarian cancer cells.

Considering the therapeutic effect of ropivacaine on ovarian cancer, this research further investigated the possible mechanism of ropivacaine protecting ovarian cancer through various molecular techniques, providing novel targets for ovarian cancer treatment in the future.

Materials and methods

Cell culture and treatment

Human ovarian cancer cell lines SKOV3 and OVCAR-3 were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in RPMI-1640 medium (Gibco, New York, USA) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO₂.

SKOV3 and OVCAR-3 cells were treated with 0, 2.5, 5, 10, 20, 40, 80 and 160 μM doses of ropivacaine (BioChemPartner, Shanghai, China) for 24 h.¹⁵ SKOV3 and OVCAR-3 cells were treated with 40 μM ropivacaine and/or 5 mmol/l erastin (MedChemExpress, New Jersey, USA),¹⁶ 40 μM ropivacaine and/or 5 μM ferrostatin-1 (MedChemExpress) for 24 h.¹⁷ SKOV3 cells were treated with 40 μM ropivacaine and/or 20 μM 740 Y-P (MedChemExpress) for 24 h.¹⁸

Cell counting kit-8 (CCK-8) assay

SKOV3 and OVCAR-3 cells (2 × 10³) were inoculated in 96-well plates. After 24 h of culture, 10 μl CCK-8 solution (Beyotime, Shanghai, China) was added to each well and placed in an incubator for further culture for 1 h. Afterward, the 96-well plates were placed in a microplate reader (Thermo Fisher Scientific) and the absorbance (OD) at 450 nm was detected.

Flow cytometry

The ALDH positive cells were determined using an Aldefluor^TM kit (Stem Cell Technologies, Shanghai, China) referring to the reagent manufacturer’s procedure. SKOV3 and OVCAR-3 cells were incubated with AldefluorTM assay buffer containing ALDH substrate and BODIPY-Aminoacetaldehyde for 50 min at 37°C. Then the ALDH+ cells were delineated by catalyzing BODIPY-Aminoacetaldehyde to its fluorescent product BODIPY-Aminoacetate; the ALDH enzymatic activity was blocked by the specific ALDH inhibitor: AldefluorTM DEAB reagent. Ultimately, the ALDH positive cells were assessed using a flow cytometer (FACSCalibur, BD Biosciences, San Jose, USA) and the BD FACSAria II System (BD Biosciences).

Sphere-formation assay

SKOV3 and OVCAR-3 (1 × 10⁴) were inoculated in 96-well ultra-low attachment plates (Corning, NY, USA) and cultured in serum-free DMEM/F12 with the addition of 20 ng/mL human recombinant epidermal growth factor (Invitrogen, Carlsbad, USA), 10 ng/mL human recombinant basic fibroblast growth factor (Invitrogen) and antibiotics penicillin and streptomycin (Thermo Fisher Scientific) for 7 days. Then the sphere number was counted under a light microscope (Olympus, Tokyo, Japan) and Image J software (National Institutes of Health, Bethesda, USA).

Western blot

After SKOV3 and OVCAR-3 cells were harvested, the total proteins were extracted from the cells by RIPA buffer (Thermo Fisher Scientific). The protein concentrations were quantified using a BCA Assay Kit (Abcam, Cambridge, UK). The total amount of proteins (30 μg) were subjected to sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific) and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, USA). After the membranes were blocked using 5% skim milk for 1 h, the membranes were incubated with the primary antibodies containing anti-OCT4 (Abcam, 1: 10,000, ab200834), anti-Nanog (Abcam, 1: 1000, ab109250), anti-GPX4 (Abcam, 1 µg/ml, ab41787), anti-SLC7A11 (xCT, Abcam, 1: 2000, ab175186), anti-p-PI3K (Abcam, 1: 500, ab182651), anti-PI3K (Abcam, 1: 1000, ab32089), anti-p-AKT (S473, Abcam, 1: 5000, ab81283), anti-p-AKT (T308, Abcam, 1: 500, ab38449), anti-AKT (Sigma-Aldrich, 1: 1000, SAB4500803), anti-p-mTOR (S2448, Abcam, 1: 1000, ab109268), anti-mTOR (Abcam, 1: 10,000, ab134,903) and anti-β-actin (Abcam, 1 µg/ml, ab8226) at 4°C overnight. The membranes were further incubated with the secondary antibody (Abcam) at room temperature for 1.5 h. The protein bands were assessed using enhanced chemiluminescence reagents (Millipore) and Image J software (National Institutes of Health).

Detection of the Fe²⁺ levels

The Fe²⁺ levels in ovarian cancer cells with different treatments were measured using an Iron Ion Kit (Millipore) following the reagent manufacturer’s procedure. In brief, the SKOV3 and OVCAR-3 cell supernatant was collected and incubated with an iron reducer, and then labeled with an iron probe. After that, an OD value of 590 nm was measured using a microplate reader (Thermo Fisher Scientific).

Immunofluorescence assay

The SKOV3 and OVCAR-3 cells with different treatments were harvested, washed with D-Hank’s buffer, and incubated with 2’,7’-dichlorofluorescein diacetate (DCFH-DA) at 37°C for 25 min. The DCF fluorescence of cells was analyzed using a fluorospectrophotometer at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The fluorescent intensity of DCF was tested by flow cytometry.

In vivo assay

Twelve Nude female BALB/c-nu mice (5-weeks-old) were from Shanghai Lab. Animal Research Center (Shanghai, China). All mice were kept in a temperature and humidity-controlled rooms with the 12 h light/dark cycle. All animal experiments were approved by the Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University.

SKOV3 cells (5 × 10⁶) were injected subcutaneously into mice according to the previously described methods with minor changes.¹⁹ To evaluate the effect of ropivacaine on the growth of ovarian cancer, ropivacaine (10 mg/kg) was injected intraperitoneally into mice referring to the previously reported methods with minor revisions.¹⁵ The size of the tumor was measured every day and the tumor volumes were calculated by the formula: length × width²/2 = tumor volume (mm³). When the tumor size reached 2000 mm³, all mice were sacrificed and the excised tumor tissues were weighed to evaluate the antitumor effect.

Immunohistochemical assay

The xenografted tumor tissues were fixed with 10% formalin (Millipore) for 24 h, dehydrated and embedded in paraffin, and then made into 5 μm sections. The expressions of Ki-67, OCT4, Nanog, GPX4 and SLC7A11 were assessed using immunohistochemistry (IHC) methods with anti-Ki-67 (Abcam, 1 µg/ml, ab15580), anti-OCT4 (Abcam, 1: 250, ab200834), anti-Nanog (Abcam, 1: 100, ab109250), anti-GPX4 (Abcam, 1: 100, ab125066) and anti-SLC7A11 (Abcam, 1 µg/ml, ab37185) antibodies. Briefly, the sections were blocked by 1% BSA and were incubated with the above primary antibodies overnight at 4°C. Followed by the incubation with a secondary antibody for 1.5 h at room temperature. Subsequently, the sections were dyed with diaminobenzidine (DAB) and counterstained with hematoxylin. Ultimately, the sections were examined under a microscope (Olympus) and the percentage of positive cells was calculated.

Statistical analysis

All data were analyzed using SPSS 20.0 software (IBM, Chicago, USA). Data were presented as mean ± standard deviation. For the IC50 value of ropivacaine, the growth curve of ovarian cancer cells was drawn with different concentrations of ropivacaine as abscissa and cell viability (%) as ordinate. The IC50 value was the ropivacaine concentration of ovarian cancer cells at 50% viability. Differences between the two groups or more than two groups were assessed by Unpaired Student’s t-test or one-way ANOVA followed by Tukey’s post-test. p < .05 was considered statistically significant.

Results

Ropivacaine restrains the ovarian cancer cell growth

The ovarian cancer cell growth was assessed after the SKOV3 and OVCAR-3 cells were treated with different doses of ropivacaine. The results demonstrated that the SKOV3 cell growth began to be repressed at a 10 μM dose of ropivacaine in comparison with the control group, and the IC50 was 18.64 μM (Figure 1(a)). Meanwhile, the growth of OVCAR-3 cells was also inhibited from the 10 μM dose of ropivacaine compared with the control group, and the IC50 was 24.86 μM ((Figure 1(b)). The 10, 20, and 40 μM doses of ropivacaine were selected for subsequent experiments referring to the experimental data of the CCK-8 assay. In general, ropivacaine repressed ovarian cancer cell growth.

Figure 1.

Effect of ropivacaine on ovarian cancer cell growth. Ovarian cancer cells SKOV3 and OVCAR-3 were treated with different concentrations (0, 2.5, 5, 10, 20, 40, 80, and 160 μM) of ropivacaine for 24 h. (A–B) The growth of SKOV3 and OVCAR-3 was evaluated by a Cell Counting Kit-8 (CCK-8) assay. *p < .05, **p < 0.01 vs. control (0 μM). The independent assays were repeated three times. Rop: ropivacaine.

Ropivacaine represses the stemness of ovarian cancer cells

To further investigate the ropivacaine function on the ovarian cancer cell stemness, the ALDH positive cells were assessed after the ovarian cancer cells were treated with different concentrations of ropivacaine. As displayed in Figure 2(a), the ALDH positive cells were decreased gradually with the increased ropivacaine concentration, and the ALDH positive cells were reduced by nearly half at the 20 μM dose. As we all know, the sphere-formation ability is a pivotal method for the identification of tumor stem cells in vitro. Furthermore, the results of the sphere-formation assay confirmed that the sphere-formation ability of SKOV3 cells was weakened from the 10 μM dose of ropivacaine, and the sphere-formation ability of OVCAR-3 cells began to be reduced at a 20 μM dose of ropivacaine (Figure 2(b)). As presented in Figure 2(c), the protein levels of markers for stem cells OCT4 and Nanog protein levels began to be decreased in SKOV3 cells at a 10 μM dose of ropivacaine, and the OCT4 and Nanog protein levels in OVCAR-3 cells were down-regulated from the 20 μM dose of ropivacaine. The protein quantification results were displayed in Figure 2(c). To sum up, ropivacaine restrained the ovarian cancer cell stemness.

Figure 2.

Influence of ropivacaine on the stemness of ovarian cancer cells. Ovarian cancer cells SKOV3 and OVCAR-3 were treated with different contents (0, 10, 20, and 40 μM) of ropivacaine for 24 h. (A) The Aldehyde dehydrogenase (ALDH) positive cells were determined by flow cytometry. (B) The sphere-formation ability of ovarian cancer cells was analyzed using a sphere-formation assay. (C) The protein levels of OCT4 and Nanog were measured by Western blot. *p < .05, **p < .01 vs. control (0 μM). The independent assays were repeated three times.

Ropivacaine accelerates ferroptosis in ovarian cancer cells

Considering the importance of Fe²⁺ levels in ferroptosis (the novel mechanisms that regulate cell death and repress ovarian cancer cell growth), the Fe²⁺ levels were assessed in SKOV3 and OVCAR-3 cells and the results demonstrated that the Fe²⁺ levels were gradually elevated with the increased ropivacaine concentration and the Fe²⁺ levels increased nearly twofold when the ropivacaine dose was 20 μM (Figure 3(a)). The SKOV3 and OVCAR-3 cells were treated with ropivacaine and/or erastin (an agonist of ferroptosis), ropivacaine and/or ferrostatin-1 (inhibitor of ferroptosis). As displayed in Figure 3(b), the Fe²⁺ levels were further elevated after the erastin treatment, while the ferrostatin-1 treatment decreased the Fe²⁺ levels. Meanwhile, the ROS levels of SKOV3 cells were elevated from the 10 μM dose of ropivacaine, and the ROS levels of OVCAR-3 cells began to be increased at a 20 μM dose of ropivacaine (Figure 3(c)). Western blot data indicated that the protein levels of ferroptosis inhibitors GPX4 and SLC7A11 began to be decreased in SKOV3 cells at a 10 μM dose of ropivacaine, and the GPX4 and SLC7A11 protein levels in OVCAR-3 cells were reduced from the 20 μM dose of ropivacaine (Figure 3(d)). The above data suggested that ropivacaine accelerated the ferroptosis in ovarian cancer cells.

Figure 3.

Regulation of ropivacaine on ferroptosis in ovarian cancer cells. (A) Ovarian cancer cells SKOV3 and OVCAR-3 were treated with different contents (0, 10, 20, and 40 μM) of ropivacaine for 24 h. The Fe²⁺ levels were detected by the Iron ion kit. (B) SKOV3 and OVCAR-3 cells were treated with 40 μM ropivacaine and/or 5 mmol/l erastin, 40 μM ropivacaine and/or 5 μM ferrostatin-1 for 24 h. Analysis of the Fe²⁺ levels using an Iron ion kit. (C) Ovarian cancer cells SKOV3 and OVCAR-3 were treated with different contents (0, 10, 20, and 40 μM) of ropivacaine for 24 h. The ROS levels of ovarian cancer cells were assessed by immunofluorescence assay (scale bar, 50 μM). (D) The GPX4 and SLC7A11 protein levels were measured using Western blot. *p < .05, **p < .01 vs. control (0 μM) or 40 μM ropivacaine. The independent assays were repeated three times.

Ropivacaine inactivates the PI3K/AKT signaling pathway in ovarian cancer cells

As reported, the abnormal activation of the PI3K/AKT signaling pathway accelerates ovarian cancer development.²⁰ Therefore, we attempted to clarify whether ropivacaine participated in regulating the activation of the PI3K/AKT signaling pathway in ovarian cancer. Total-PI3K, total-AKT, and total-mTOR were internal controls for the calculation of relative expressions of p-PI3K, p-AKT (S473), p-AKT (T308) and p-mTOR (S2448), respectively. As displayed in Figure 4(a), the protein levels of p-PI3K and p-AKT (S473), p-AKT (T308), and p-mTOR (S2448) in cells were gradually decreased with the increase of ropivacaine concentration, while the total-PI3K, total-AKT and total-mTOR protein levels did not change significantly. After the SKOV3 cells were treated with ropivacaine and/or 740 Y-P (a commonly used PI3K agonist), the protein levels of p-PI3K and p-AKT (S473), p-AKT (T308), and p-mTOR (S2448) in cells were down-regulated after the ropivacaine treatment, while the 740 Y-P treatment partially reversed this down-regulation; the total-PI3K, total-AKT, and total-mTOR protein levels were unchanged between all groups (Figure 4(b)). Meanwhile, the CCK-8 results corroborated that the ropivacaine treatment reduced cell viability, and this reduction was partially reversed by the 740 Y-P treatment (Figure 4(c)). As displayed in Figure 4(d), the sphere-formation ability of ovarian cancer cells was weakened after the ropivacaine treatment, and this trend was partially reversed by the 740 Y-P treatment. Furthermore, the treatment of ropivacaine increased the Fe²⁺ levels, and the Fe²⁺ levels were increased nearly threefold, while the treatment of 740 Y-P partially reversed this increase (Figure 4(e)). Similarly, the ROS levels of ovarian cancer cells were elevated after the ropivacaine treatment, and this effect was partially reversed by the 740 Y-P treatment (Figure 4(f)). In summary, ropivacaine inactivated the PI3K/AKT signaling pathway in ovarian cancer cells.

Figure 4.

Effect of ropivacaine on the PI3K/AKT signaling pathway in ovarian cancer cells. (A) Ovarian cancer cells SKOV3 were treated with different contents (0, 10, 20, and 40 μM) of ropivacaine for 24 h. Analysis of the p-PI3K, total-PI3K, p-AKT (S473), p-AKT (T308), total-AKT, p-mTOR (S2448), and total-mTOR protein levels in cells by Western blot. (B) The SKOV3 cells were treated with 40 μM ropivacaine and/or 20 μM 740 Y-P for 24 h. Detection of the p-PI3K, total-PI3K, p-AKT (S473), p-AKT (T308), total-AKT, p-mTOR (S2448), and total-mTOR protein levels in cells using Western blot. (C) The viability of SKOV3 cells was assessed by CCK-8 assay. (D) The sphere-formation ability of SKOV3 cells was evaluated using a sphere-formation assay. (E) The Fe²⁺ levels were measured by an Iron ion kit. (F) Analysis of the ROS levels of SKOV3 cells using immunofluorescence assay (scale bar, 50 μM). *p < .05, **p < .01 vs 0 μM ropivacaine, control or 40 μM ropivacaine. Con: control. The independent assays were repeated three times.

Ropivacaine restrains the proliferation of ovarian cancer cells in vivo

Subsequently, we further evaluated the function of ropivacaine in ovarian cancer in vivo. The ropivacaine treatment reduced the tumor volume in comparison with the sham group (Figure 5(a)) and the representative macroscopic figures of tumors were provided in Supplementary Figure 1. Also, the treatment of ropivacaine had no significant changes in the weight of mice in comparison with the sham group (Figure 5(b)). Meanwhile, the ropivacaine treatment decreased the tumor weight compared with the sham group (Figure 5(c)). Besides, the expressions of Ki-67 (proliferating marker), OCT4, Nanog, GPX4, and SLC7A11 in subcutaneous tumor tissues of mice were measured and the results confirmed that the ropivacaine treatment reduced the Ki-67, OCT4, Nanog, GPX4 and SLC7A11 expressions in tissues (Figure 5(d)). The above data suggested that ropivacaine weakened the proliferation of ovarian cancer cells in vivo.

Figure 5.

Ropivacaine regulates the proliferation of ovarian cancer cells in vivo. SKOV3 cells (5 × 10⁶) were injected subcutaneously into mice and ropivacaine (10 mg/kg) was injected intraperitoneally into mice. (A–C) Analysis of the tumor volume, mice weight, and tumor weight between different groups. (D) The expressions of Ki-67 (proliferating marker), OCT4, Nanog, GPX4, and SLC7A11 in subcutaneous tumor tissues of mice were assessed using an immunohistochemical assay (scale bar, 50 μM). **p < .01 vs. sham. The independent assays were repeated three times.

Discussion

Ovarian cancer seriously affects the physical and mental health of women, so it is urgent to explore effective treatment strategies.²¹ Ropivacaine has an antitumor role in various human tumors.²² However, the ropivacaine role in ovarian cancer remained uncertain. In this study, we treated ovarian cancer cell lines with different doses of ropivacaine and demonstrated that ropivacaine inhibited ovarian cancer cell growth, implying that ropivacaine might exert a protective role in ovarian cancer. This study is the first to investigate the ropivacaine function in ovarian cancer.

Recently, the important function of cancer stem cells in ovarian cancer has been widely recognized with the deepening of research. LncRNA HOX transcript antisense intergenic RNA maintains the stemness of ovarian cancer stem cells through regulating the miR-206/TBX3 axis²³; platelet-activating factor accelerates the spheroid formation and increases the stemness genes expressions, thus aggravating ovarian cancer,²⁴ which implies that reducing the ovarian cancer cell stemness is expected to relieve ovarian cancer. Considering that cell subsets with high ALDH activity in tumors are closely related to the stemness of cancer stem cells,²⁵ this study further confirmed that the ALDH positive cells were decreased gradually with the increased ropivacaine concentration. Also, we demonstrated that the ropivacaine treatment repressed the ovarian cancer cell stemness by sphere-formation assay. Meanwhile, as the markers for stemness, OCT4 and Nanog are commonly used to evaluate the stemness of tumor cells.²⁶ This study further indicated that ropivacaine down-regulated the OCT4 and Nanog protein levels. The above findings revealed that ropivacaine repressed the ovarian cancer cell stemness.

Ferroptosis is a novel cell death pathway first proposed by Dixon et al²⁷ and is characterized by the accumulation of iron-dependent lipid peroxides.²⁸ Various studies have been conducted on the mechanism of ferroptosis since the concept of ferroptosis is proposed. To date, several regulatory mechanisms and signaling pathways of ferroptosis have been identified. For instance, Zhang et al. confirmed that the attenuation of the PKCβII-ACSL4 pathway reduces the accumulation of lipid peroxidation, thereby effectively blocking ferroptosis²⁹; Yi et al. indicated that the abnormal activation of the PI3K-AKT-mTOR signaling pathway protects cancer cells from oxidative stress and ferroptosis through SREBP1/SCD1-mediated lipogenesis.³⁰ In recent years, the ferroptosis function in ovarian cancer has been gradually confirmed. As reported, ferroptosis promotes the occurrence and development of ovarian cancer and that ferroptosis might aggravate ovarian cancer by mediating tumor metastasis.³¹ A recent study reveals that erastin and cisplatin induce ferroptosis by increasing the level of reactive oxygen species, thereby inhibiting ovarian cancer cell growth and alleviating ovarian cancer.³² Considering the inhibitory effect of ropivacaine on ovarian cancer cell growth, we attempted to clarify whether ropivacaine mediated the ferroptosis in ovarian cancer cells. As expected, our data revealed that the different doses of ropivacaine facilitated ferroptosis in ovarian cancer cells, which was similar to the above conclusions.

In recent decades, the molecular mechanisms of various genetic factors in cancer have been revealed, including Phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT).³³ PI3K is a lipid kinase and an important cause of chemotherapy resistance in cancer treatment.³⁴ AKT is a critical downstream effector of the PI3K signaling pathway and regulates various pathways, including restraining cell apoptosis, stimulating cell growth, and regulating cell metabolism.³⁵ Accumulated studies corroborate that the PI3K/AKT signaling pathway is involved in the regulation of the occurrence and development of multiple tumors. For example, the carcinogenic function of IGF2BP2 in pancreatic cancer is achieved through the activation of the PI3K/AKT signaling pathway to accelerate tumor proliferation³⁶; microRNA functions in the growth, and metastasis of breast cancer cells by targeting the PI3K/AKT carcinogenic signaling pathway.³⁷ Emerging evidence suggests that the PI3K/AKT signaling pathway participates in ovarian cancer progression. Ediriweera et al. confirmed the importance of the PI3K/AKT signaling pathway in ovarian cancer progression and comprehensively analyze the clinical application of the PI3K/AKT signaling pathway inhibitors in ovarian cancer³⁸; Deng et al. expounded that inactivating the PI3K/AKT signaling pathway reduces ovarian cancer chemoresistance by decreasing the cancer stem cell markers levels,³⁹ implying that repressing the abnormal activation of the PI3K/AKT signaling pathway slowed down the occurrence and development of ovarian cancer. Based on our previous experimental results that ropivacaine repressed the ovarian cancer cell growth, we attempted to clarify whether ropivacaine mediated the ovarian cancer progression through the PI3K/AKT signaling pathway. Interestingly, our results confirmed that the ropivacaine treatment inactivated the PI3K/AKT signaling pathway in ovarian cancer cells. After confirming the function and molecular mechanism of ropivacaine in ovarian cancer in vitro, we further verified the protective function of ropivacaine in ovarian cancer in vivo. As expected, ropivacaine reduced the proliferation of ovarian cancer cells in vivo, which was similar to the in vitro results.

In conclusion, we confirmed the ropivacaine function in ovarian cancer from the following multiple perspectives: (1) ropivacaine treatment repressed the ovarian cancer cell growth; (2) ropivacaine inhibited the stemness of ovarian cancer cells; (3) ropivacaine accelerated the ferroptosis in ovarian cancer cells. After confirming that ropivacaine had a protective function against ovarian cancer, we further investigated its potential molecular mechanism. Our data corroborated that ropivacaine inactivated the PI3K/AKT signaling pathway in ovarian cancer cells. Meanwhile, the in vivo assay indicated that ropivacaine restrained the proliferation of ovarian cancer cells in vivo. This research might provide novel drugs and potential biomarkers for ovarian cancer treatment. Furthermore, a recent research has indicated that Frizzled-7 restrains ferroptosis and promotes stemness both in vitro and in vivo models of ovarian cancer, thereby exacerbating ovarian cancer.⁴⁰ Thus, the limitation of this study mainly lies in that whether ropivacaine repressed the proliferation of ovarian cancer cells by activating ferroptosis was not investigated in vivo. We would further explore in vivo in the future to enrich the content of this study. Moreover, in view of the important role of ropivacaine in ovarian cancer models in vivo and in vitro, we would verify the ropivacaine function in clinical tissue samples to further enrich the content of this study.

Supplemental Material

Supplemental Material - Ropivacaine represses the ovarian cancer cell stemness and facilitates cell ferroptosis through inactivating the PI3K/AKT signaling pathway

Supplemental Material for Ropivacaine represses the ovarian cancer cell stemness and facilitates cell ferroptosis through inactivating the PI3K/AKT signaling pathway by Yi Lu, Jinbao Mao, Yanbing Xu, Hao Pan, Yu Wang and Wei Li in Human & Experimental Toxicology

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Wei Li

Supplemental Material

Supplemental material for this article is available online.

References

Zhao

Liu

, et al. The expression of cancer-testis antigen in ovarian cancer and the development of immunotherapy. Am Journal Cancer Research 2022; 12: 681–694.

Yang

, et al. Elevated LINC00909 Promotes Tumor Progression of Ovarian Cancer via Regulating the miR-23b-3p/MRC2 Axis. Oxidative Medicine Cellular Longevity 2021; 2021: 5574130. DOI: 10.1155/2021/5574130.

Chilimoniuk

Rocka

Stefaniak

, et al. Molecular methods for increasing the effectiveness of ovarian cancer treatment: a systematic review. Future Oncology (London, England) 2022; 18: 1627–1650. DOI: 10.2217/fon-2021-0565.

Liu

Zhang

Song

, et al. Lipid metabolism of cancer stem cells. Oncol Letters 2022; 23: 119. DOI: 10.3892/ol.2022.13239.

Wilczyński

Paradowska

. Cancer stem cells in ovarian cancer-A source of tumor success and a challenging target for novel therapies. Int Journal Molecular Sciences 2022; 23: 2022. DOI: 10.3390/ijms23052496

Muñoz-Galván

Felipe-Abrio

Verdugo-Sivianes

, et al. Downregulation of MYPT1 increases tumor resistance in ovarian cancer by targeting the Hippo pathway and increasing the stemness. Mol Cancer 2020; 19: 7. DOI: 10.1186/s12943-020-1130-z.

Chen

Jiang

, et al. Targetting ferroptosis for blood cell-related diseases. J Drug Targeting 2022; 30: 244–258. DOI: 10.1080/1061186x.2021.1971237.

Zhao

Zhou

, et al. Ferroptosis: opportunities and challenges in myocardial ischemia-reperfusion injury. Oxidative Medicine Cellular Longevity 2021; 2021: 9929687. DOI: 10.1155/2021/9929687.

Chan

Yung

Chan

, et al. MAP30 protein from momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis. Pharmacol Research 2020; 161: 105157. DOI: 10.1016/j.phrs.2020.105157.

10.

Dregalla

Uribe

Bodor

. Effect of local anesthetics on platelet physiology and function. J Orthopaedic Research 2021; 39: 2744–2754. DOI: 10.1002/jor.25019

11.

Chen

Liu

Guo

, et al. Ropivacaine inhibits cervical cancer cell growth via suppression of the miR-96/MEG2/pSTAT3 axis. Oncol Reports 2020; 43: 1659–1668. DOI: 10.3892/or.2020.7521.

12.

Zhang

Peng

Zheng

. Ropivacaine inhibits the migration of esophageal cancer cells via sodium-channel-independent but prenylation-dependent inhibition of Rac1/JNK/paxillin/FAK. Biochem Biophysical Research Communications 2018; 501: 1074–1079. DOI: 10.1016/j.bbrc.2018.05.110.

13.

Nie

Cai

. SNAT2/SLC38A2 confers the stemness of gastric cancer cells via regulating glutamine level. Dig Diseases Sciences 2021; 67(7): 2948–2956. DOI: 10.1007/s10620-021-07110-2.

14.

Jia

Zhou

, et al. Tanshinone IIA attenuates the stemness of breast cancer cells via targeting the miR-125b/STARD13 axis. Exp Hematology Oncology 2022; 11: 2. DOI: 10.1186/s40164-022-00255-4

15.

Zhang

Xing

, et al. Ropivacaine inhibits the growth, migration and invasion of gastric cancer through attenuation of WEE1 and PI3K/AKT signaling via miR-520a-3p. OncoTargets Therapy 2020; 13: 5309–5321. DOI: 10.2147/ott.S244550.

16.

Sun

Zhang

. Lidocaine promoted ferroptosis by targeting miR-382-5p/SLC7A11 axis in ovarian and breast cancer. Front Pharmacology 2021; 12: 681223. DOI: 10.3389/fphar.2021.681223.

17.

Zhou

Chen

Cai

, et al. Erastin reverses ABCB1-mediated docetaxel resistance in ovarian cancer. Front Oncology 2019; 9: 1398. DOI: 10.3389/fonc.2019.01398.

18.

Zhang

, et al. Tivantinib hampers the proliferation of glioblastoma cells via PI3K/Akt/Mammalian target of rapamycin (mTOR) signaling. Med Science Monitor: International Medical Journal Experimental Clinical Research 2019; 25: 7383–7390. DOI: 10.12659/msm.919319

19.

Yuan

Qian

Guo

, et al. LncRNA-ATB promotes the tumorigenesis of ovarian cancer via targeting miR-204-3p. OncoTargets Therapy 2020; 13: 573–583. DOI: 10.2147/ott.S230552.

20.

Lin

Zhou

, et al. NOL4L, a novel nuclear protein, promotes cell proliferation and metastasis by enhancing the PI3K/AKT pathway in ovarian cancer. Biochem Biophysical Research Communications 2021; 559: 121–128. DOI: 10.1016/j.bbrc.2021.04.055.

21.

Croft

Sharma

Godbole

, et al. Ovarian-cancer-associated extracellular vesicles: microenvironmental regulation and potential clinical applications. Cells 2021; 10(9): 2272. DOI: 10.3390/cells10092272

22.

Zhang

Tan

et al. Local anesthetic ropivacaine exhibits therapeutic effects in cancers. Front Oncology 2022; 12(Published online 2022 Feb 3): 836882, doi:10.3389/fonc.2022.836882, PMID: 35186766.

23.

Zhang

Guo

Cai

, et al. HOTAIR maintains the stemness of ovarian cancer stem cells via the miR-206/TBX3 axis. Exp Cell Research 2020; 395: 112218. DOI: 10.1016/j.yexcr.2020.112218.

24.

Gao

Zhao

Yao

, et al. Platelet-activating factor induces the stemness of ovarian cancer cells via the PAF/PAFR signaling pathway. Am Journal Translational Research 2020; 12: 7249–7261.

25.

Tsochantaridis

Voulgaridou

Giatromanolaki

, et al. Profiling of aldehyde dehydrogenase isoforms in in vitro formed tumorspheres. Anticancer Research 2021; 41: 5481–5488. DOI: 10.21873/anticanres.15361.

26.

Chang

Zhang

Huo

, et al. Substrate rigidity dictates colorectal tumorigenic cell stemness and metastasis via CRAD-dependent mechanotransduction. Cel Reports 2022; 38: 110390. DOI: 10.1016/j.celrep.2022.110390.

27.

Dixon

. Ferroptosis: bug or feature? Immunological Reviews 2017; 277: 150–157. DOI: 10.1111/imr.12533.

28.

Lei

Zhuang

Gan

. Targeting ferroptosis as a vulnerability in cancer. Nat Reviews Cancer 2022; 22(7): 381–396. DOI: 10.1038/s41568-022-00459-0.

29.

Zhang

, et al. PKCβII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis. Nat Cell Biology 2022; 24: 88–98. DOI: 10.1038/s41556-021-00818-3.

30.

Zhu

, et al. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proc Natl Acad Sci United States America 2020; 117: 31189–31197. DOI: 10.1073/pnas.2017152117.

31.

You

Fan

Huang

, et al. Ferroptosis-related gene signature promotes ovarian cancer by influencing immune infiltration and invasion. J Oncology 2021; 2021: 9915312. DOI: 10.1155/2021/9915312.

32.

Cheng

Bao

, et al. Erastin synergizes with cisplatin via ferroptosis to inhibit ovarian cancer growth in vitro and in vivo. Journal Obstetrics Gynaecology Research 2021; 47: 2481–2491. DOI: 10.1111/jog.14779.

33.

Khezri

Jafari

Yousefi

, et al. The PI3K/AKT signaling pathway in cancer: molecular mechanisms and possible therapeutic interventions. Exp Molecular Pathology 2022; 2022: 104787. DOI: 10.1016/j.yexmp.2022.104787.

34.

Meng

Zhang

, et al. Development of PI3K inhibitors: advances in clinical trials and new strategies (Review). Pharmacol Research 2021; 173: 105900. DOI: 10.1016/j.phrs.2021.105900.

35.

Zhang

Xuan

Huang

, et al. Dictamnine inhibits pancreatic cancer cell growth and epithelial-mesenchymal transition by blocking the PI3K/AKT signaling pathway. Neoplasma 2022; 69(3): 603–619. DOI: 10.4149/neo_2022_211016N1474.

36.

Zong

, et al. Up-regulation of IGF2BP2 by multiple mechanisms in pancreatic cancer promotes cancer proliferation by activating the PI3K/Akt signaling pathway. J Experimental Clinical Cancer Research : CR 2019; 38: 497. DOI: 10.1186/s13046-019-1470-y.

37.

Tao

, et al. Breast cancer cells-derived von willebrand factor promotes VEGF-A-related angiogenesis through PI3K/Akt-miR-205-5p signaling pathway. Toxicol Applied Pharmacology 2022; 440: 115927. DOI: 10.1016/j.taap.2022.115927.

38.

Ediriweera

Tennekoon

Samarakoon

. Role of the PI3K/AKT/mTOR signaling pathway in ovarian cancer: Biological and therapeutic significance. Semin Cancer Biology 2019; 59: 147–160. DOI: 10.1016/j.semcancer.2019.05.012.

39.

Deng

Bai

Feng

, et al. Inhibition of PI3K/Akt/mTOR signaling pathway alleviates ovarian cancer chemoresistance through reversing epithelial-mesenchymal transition and decreasing cancer stem cell marker expression. BMC Cancer 2019; 19: 618–2019. DOI: 10.1186/s12885-019-5824-9.

40.

Wang

Zhao

Condello

, et al. Frizzled-7 identifies platinum-tolerant ovarian cancer cells susceptible to ferroptosis. Cancer Research 2021; 81: 384–399. DOI: 10.1158/0008-5472.Can-20-1488.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.17 MB

Ropivacaine represses the ovarian cancer cell stemness and facilitates cell ferroptosis through inactivating the PI3K/AKT signaling pathway

Abstract

Purpose

Materials and methods

Results

Conclusion

Keywords

Introduction

Materials and methods

Cell culture and treatment

Cell counting kit-8 (CCK-8) assay

Flow cytometry

Sphere-formation assay

Western blot

Detection of the Fe2+ levels

Immunofluorescence assay

In vivo assay

Immunohistochemical assay

Statistical analysis

Results

Ropivacaine restrains the ovarian cancer cell growth

Ropivacaine represses the stemness of ovarian cancer cells

Ropivacaine accelerates ferroptosis in ovarian cancer cells

Ropivacaine inactivates the PI3K/AKT signaling pathway in ovarian cancer cells

Ropivacaine restrains the proliferation of ovarian cancer cells in vivo

Discussion

Supplemental Material

Supplemental Material - Ropivacaine represses the ovarian cancer cell stemness and facilitates cell ferroptosis through inactivating the PI3K/AKT signaling pathway

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

Supplemental Material

References

Supplementary Material

Detection of the Fe²⁺ levels